This invention relates generally to the detection of urinary trypsin inhibitors (UTIs) in human urine. UTIs inhibit one or more of the Serine proteases. Trypsin is a member of the family of Serine proteases, i.e. enzymes, that includes trypsin, elastase, kallikrein, plasmin, thrombin, chymotrypsin, and cathepsin, among others. This group of inhibitors primarily forms after an increase in the number of white blood cells in the body due to the release of elastase during infection or inflammation. UTIs are not normally found in the urine produced by healthy individuals. The amount is elevated in those whose bodies have bacterial infections and inflammatory disorders or other maladies such as malignant tumors, kidney disease, myocardial infarction, lung emphysema, surgical trauma, and kidney stones among others.
When infections and/or inflammation occur, the bodies' response involves the production of serine proteases such as elastase released by neutrophils. Non-inhibitor forms of UTI, called pro-inhibitors, such as interleukin-α-inhibitor (I-α-I) and the pre-interleukin-α-inhibitor (P-α-I), circulate freely in plasma of healthy and diseased individuals. Serine proteases cause proteolysis of the pro-inhibitors and release the lower molecular weight UTIs into active function. The released urinary trypsin inhibitors act on serine proteases and are later excreted in the urine. Discovered in 1909, urine trypsin inhibitors are Kunitz-type protease inhibitors and have been named HI-30, Mingin, Urinastatin, Serpin, and Ulinastatin over the years with the scientific community settling on the name Bikunin for a prevalent fragment of ˜0.30 Kda molecular weight. The amino acid sequence of the Bikunin inhibitor fragment is known. It contains two Kunitz inhibitory binding domains and a large and variable chondroitin sulfate chain. See the International Journal of Biochemistry and Cell Biology 32(2000) 125-137.
Although the amount of urinary trypsin inhibitors has been measured by several methods, e.g. enzyme inhibition, antibody stains, latex agglutination, and radioimmunoassays, all of the UTIs in the sample are measured. It has not been shown that certain forms of the inhibitor can result from chronic disease and are more easily distinguished from the inhibitors which were present in individuals with lesser degrees of inflammation. As a result, the diagnostic use of UTIs has to date only been as a non-specific marker of infection and/or inflammation. The non-specific nature lessens the clinical utility as determination of UTIs does not help the care giver to know the type or site of infection and makes it difficult to separate conditions for which the patient would need therapy from general ailments. The present invention is directed toward making such distinctions.
One method of measuring the UTI content of a urine sample involves the addition to the sample of known amounts of trypsin and then measuring the degree to which the trypsin has been inhibited. Examples of this technique can be found in three published U.S. Patent Applications 2001/0055816 A1, 2002/0004219 A1, and 2003/0125577 A1. In these published applications, a known amount of trypsin on a substrate capable of producing a detectable response is added to a sample of urine. The substrate is cleaved by trypsin to yield detectable byproducts. If trypsin inhibitors are present, the response is diminished since some of the available substrate is not cleaved. Thus, by measuring the amount of the trypsin present and functioning relative to the amount added, the UTI content can be determined. This method detects any inhibitor of trypsin activity and is non-specific for any given trypsin inhibitor excreted into urine. The patent application published in 2001 discloses the use of a polycarboxylic chelating agent to inhibit the interference of calcium in the sample. The patent applications published in 2002 and 2003 concern certain aromatic esters of arginine shown to be useful as substrates for trypsin in the method just described.
Another method which determines directly the amount of UTIs in a urine sample involves the development of antibodies which attach themselves to the urinary trypsin inhibitors and which, by the addition of immunoassay reagents containing the antibody to a sample, produce a response signal. There are various immunoassay methods which could be used to apply antibodies of the invention such as microparticle capture immunoassays (MIC), latex agglutination inhibition (LAI), solid phase chromatographic (IC), radioimmunoassays (RIA), enzyme linked immunosorbent assays (ELISA), enzyme linked assays (EIA), fluorescence linked assays (FIA), luminescence linked assays (LIA), rare earth metals label assays, chemiluminescence assays (CLA) and optical color label assays (OA) such as colored latex particle and colloidal gold. It is also feasible to use electrochemical signal transducers (EST) based on amperometric, impedimetric, and potentiometric detection methods.
In principle, immunoassays can be of either a heterogeneous format requiring a separation step or a homogeneous format without separation and either of a competitive or a non-competitive nature. For heterogeneous assays, solid phases can be used to separate bound antigen from free antigen and can include plastic wells, tubes, capillaries, membranes, latex particles, and magnetic particles. Antibodies are attached to the solid phases. Antibodies can also be attached or conjugated (labeled) with reagents that directly or indirectly produce detectable responses, other antibodies or other reagents in a variety of fashions. An immunoassay can also employ multiple and different antibodies in a variety of manners such sandwich assays, double label assays, and multiple sandwich assays depending on the detection needs for the material being detected.
In general, the immunoassay reagents undergo changes whereby a signal is generated and the intensity of the signal generated is proportional to the concentration of the analyte measured in the specimen or sample. Immunoassay reagents contain indicator dyes, metals, enzymes, polymers, antibodies, surface active agents, particles, electrochemically reactive ingredients and various other chemicals dried or filled onto carriers. Carriers often used are tubes, cups, capillaries, strips, vials, papers, microfluidic devices, cassettes, membranes or polymers with various sample volume, uptake and transport properties.
It has been found that polyclonal antibodies produced from rabbits inoculated with urinary trypsin inhibitors purified from the urine of patients with kidney disease are useful in measuring the amount of total UTIs in urine samples from both healthy individuals and those with disease. However, the polyclonal antibodies are not able to distinguish the various forms of UTI from each other, in particular from the pro-inhibitors interleukin-α-inhibitor (I-α-I) and pre-interleukin-α-inhibitor (P-α-I). Since all these proteins are readily present in the healthy patients, the cross reactivity of the polyclonal antibody makes it useless for distinguishing diseased patients from healthy patients by using blood specimens. In the case of urine specimens, the high molecular weights of P-α-I and I-α-I make it less likely that they will pass through the kidney, thus the polyclonal antibodies were more effective in spite of their reduced specificity. However due to the non-specific nature of the polyclonal antibodies they have not been found to be more effective than the general enzyme inhibition method previously discussed. The polyclonal antibodies were cross-reactive to all forms of UTI in urine and not specific to any one form and would measure both inhibitory and non-inhibitory UTI.
In Journal of Immunoassay 1991; 12:347-69, Trefz et al disclose the use of a monoclonal antibody produced from mice immunized with high molecular weight (240 kDa). Inter-α-trypsin inhibitor (an inactive pro-inhibitor) in an enzyme-linked immunoassay (ELISA). The results showed that the method was effective to distinguish between healthy individuals and those with disease, since the level of UTIs in the urine of those individuals with disease was higher than those without disease. The authors noted that Inter-α-trypsin inhibitor (ITI) having a molecular weight of about 240 kDa, contained several lower molecular weight peptides, understood to result from disintegration of the ITI. Their monoclonal antibody, IATI5, was found to recognize three major bands, a 240 kDa, a 120 kDa, and a 50 kDa band. In a purified HI-30 preparation their monoclonal antibody detected a protein of about 33 kDa.
In their work reported in the Journal of Biological Chemistry, Vol. 275, No. 28, issue of July 14, pp. 21185-21191, 2000, Hirashi Kobayashi et al. compared polyclonal and monoclonal antibodies raised against a purified preparation of UTI that was shown to have molecular weight in the range of about 40-80 kDa in SDS-PAGE and Western blotting. Derivatives were found to have molecular weights of about of 7 kDa, 30 kDa, and 60 kDa.
During the cross-reactivity studies of a polyclonal antibody for UTI prepared by Bayer, we were surprised to discover that further breakdown of the inhibitory Bikunin occurs during the acute phase infections in patients leading to the formation of other inhibitory UTIs containing both Kunitz inhibitor domains, but lacking the chondroitin sulfate chain. This UTI, termed Uristatin, has a molecular weight of ˜17 kDa. In a paper (Clinical Chemistry Acta (2004) 341, 73-81) reporting tests with a dipstick for detecting urinary trypsin inhibitors, Pugia et al showed that the dipstick reported the presence of two forms for UTIs; Bikunins and Uristatins. They identified the typical molecular weight of Bikunin (30.9 kDa) and three key forms of the Uristatin, designated Uristatin-1 (5.9 kDa), Uristatin-2 (8.5 kDa) and the combination of Uristatin-1 and Uristatin-2 which was termed Uristatin (17.4 kDa). All forms of Uristatin lack the chondroitin sulfate chain, and are very prevalent in patient specimens when analyzed by electrophoresis. All Uristatin forms are inhibitory to the trypsin family of proteases; therefore they contain at one of the two Kunitz inhibitory domains that inhibit the protease active site upon binding. Uristatin-1 contains binding domain 2 and Uristatin-2 contains binding domain 1.
It was further noted that, given the conditions of the patient used for collection, the typical molecular weights of IUTIs could vary considerably. Additional variations of Bikunin and Uristatin are due to fragmentation of peptide structure, variations in the peptide sequence and variations in carbohydrate sequences attached to the Bikunin and Uristatin. Variations in molecular weight resulting from fragments occur by cleavage of the peptide sequence. A high degree of fragmentation is expected during inflammation as the inhibitors are exposed to the proteases that can cause cleavage. Elongation and fragmentation of the carbohydrate portions was also expected during inflammation as the glycoprotein are metabolized by a number of glycosyl transferases and glycosidases causing a great number of possible variants to the chondroitin sulfate chain attached to Bikunin and to the sugar side chains attached to Uristatin. Additional variations also occur by aggregation of the fragments into diners or higher oligomers, especially through association and metabolism of the carbohydrate portions. Therefore the functional UTI proteins represent a range of possible proteins around a typical molecular weight.
An additional protein in this suprafamily is the precursor protein (AMBK) that initiates the biosynthesis of the inhibitory and pro-inhibitor forms. This protein is present in the plasma of patients when the genes signal up-regulation to initiate the biosynthesis. The AMBK contains the Bikunin but lacks the heavy chains that inactivate the inhibitory domains and is attached to alpha-1-microglobulin. The detection of this form of the inhibitor is also important in determining the body's response to inflammation and infection.
There remains a need for a method of measuring specific urinary trypsin inhibitors that is able to detect the presence of disease by determining certain characteristic UTIs not found in healthy individuals. The present inventors have developed a method for making such determinations by using certain monoclonal antibodies, as will be described in detail below.